Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K ؉ (KATP) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with K ATP channel-dependent membrane excitability remains elusive. Here, we identify that the response of KATP channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the KATP channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and K ATP channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with K ATP channels and associated functions. D elivery of metabolic signals to intracellular compartments is a critical determinant of cellular homeostasis. In particular, efficient communication between cellular energetics and membrane metabolic sensors is required for regulation of cell excitability and associated functions (1, 2). Plasmalemmal ATPsensitive K ϩ (K ATP ) channels, formed by the Kir6.2 potassium channel and the sulfonylurea receptor (SUR), are unique nucleotide sensors that adjust membrane potential in response to intracellular metabolic oscillations (2-5). Transition of the SUR subunit from the ATP to the ADP-liganded state promotes K ϩ permeation through Kir6.2 and defines K ATP channel activity (5-7). However, the mechanism that facilitates nucleotide exchange in the K ATP channel environment and promotes coupling of membrane electrical events with cellular metabolic pathways remains unknown.Cellular phosphotransfer reactions catalyze nucleotide exchange facilitating communication between sites of ATP generation and ATP utilization (8-11). In this way, the phosphotransfer enzyme adenylate kinase (AK) amplifies metabolic signals and promotes intracellular phosphoryl transfer by catalyzing the reaction ATP ϩ AMP 7 2ADP (12, 13). Adenylate kinase has a distinct signaling role in setting the cellular response to stress through activation of AMP-dependent processes (12-15). Deletion of the major AK isoform (AK1) results in disturbed muscle energetic economy and decreased tolerance to metabolic stress (14, 15). Mutations in AK compromise nucleotide export from mitochondria (16), as well as the function of ATP-binding cassette transporters (17). Conversely, stimulation of AK phosphotransfer promotes nucleotide-dependent membrane functions (18,19). However, the actual significance of AK phosphotransfer in communicating energetic signals to membrane metabolic sensors, such as the K ATP cha...

Ef®cient cellular energy homeostasis is a critical determinant of muscle performance, providing evolutionary advantages responsible for species survival. Phosphotransfer reactions, which couple ATP production and utilization, are thought to play a central role in this process. Here, we provide evidence that genetic disruption of AK1-catalyzed b-phosphoryl transfer in mice decreases the potential of myo®bers to sustain nucleotide ratios despite up-regulation of high-energy phosphoryl¯ux through glycolytic, guanylate and creatine kinase phosphotransfer pathways. A maintained contractile performance of AK1-de®cient muscles was associated with higher ATP turnover rate and larger amounts of ATP consumed per contraction. Metabolic stress further aggravated the energetic cost in AK1 ±/± muscles. Thus, AK1-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy, enabling skeletal muscle to perform at the lowest metabolic cost.

Rapid exchange of high energy carrying molecules between intracellular compartments is essential in sustaining cellular energetic homeostasis. Adenylate kinase (AK)-catalyzed transfer of adenine nucleotide ␤-and ␥-phosphoryls has been implicated in intracellular energy communication and nucleotide metabolism. To demonstrate the significance of this reaction in cardiac energetics, phosphotransfer dynamics were determined by [18 O]phosphoryl oxygen analysis using 31 P NMR and mass spectrometry. In hearts with a null mutation of the AK1 gene, which encodes the major AK isoform, total AK activity and ␤-phosphoryl transfer was reduced by 94% and 36%, respectively. This was associated with up-regulation of phosphoryl flux through remaining minor AK isoforms and the glycolytic phosphotransfer enzyme, 3-phosphoglycerate kinase. In the absence of metabolic stress, deletion of AK1 did not translate into gross abnormalities in nucleotide levels, ␥-ATP turnover rate or creatine kinase-catalyzed phosphotransfer. However, under hypoxia AK1-deficient hearts, compared with the wild type, had a blunted AK-catalyzed phosphotransfer response, lowered intracellular ATP levels, increased P i /ATP ratio, and suppressed generation of adenosine. Thus, although lack of AK1 phosphotransfer can be compensated in the absence of metabolic challenge, under hypoxia AK1-knockout hearts display compromised energetics and impaired cardioprotective signaling. This study, therefore, provides first direct evidence that AK1 is essential in maintaining myocardial energetic homeostasis, in particular under metabolic stress. Adenylate kinase (AK)1 catalyzes reversible phosphotransfer, 2 ADP 7 AMP ϩ ATP, and participates in de novo synthesis, regeneration and salvage of adenine nucleotides (1-5). AK is particularly abundant in tissues with high energy turnover, where it facilitates transfer of energy-rich ␤-and ␥-phosphoryls and regulates vital ATP-dependent cellular processes (6 -10).In fact, AK may serve as an integral component of phosphotransfer networks, along with creatine kinase (CK) and glycolysis, effectively coupling ATP-generating with ATP-consuming or ATP-sensing intracellular sites (11-15).In the heart, CK-catalyzed phosphotransfer is the major pathway that can transfer high energy phosphoryls derived from the ␥-phosphoryl of ATP (10, 16 -18). Although less active than CK, AK catalysis provides a unique mechanism for transfer and utilization of both ␥-and ␤-phosphoryls in the ATP molecule (10, 15). In this way, AK-catalyzed phosphotransfer doubles the energetic potential of ATP and could provide an additional energetic source under conditions of increased energy demand (10, 19). However, due to lack of membrane permeant and selective AK inhibitors, the biological importance of AK in heart muscle and its role in sustaining myocardial energetics under conditions of metabolic stress have not been established.We have recently demonstrated that deletion of the AK1 gene, which encodes the major AK isoform, produces a phenotype with reduced skelet...

The production of AMP by adenylate kinase (AK) and subsequent deamination by AMP deaminase limits ADP accumulation during conditions of high-energy demand in skeletal muscle. The goal of this study was to investigate the consequences of AK deficiency (Ϫ/Ϫ) on adenine nucleotide management and whole muscle function at high-energy demands. To do this, we examined isometric tetanic contractile performance of the gastrocnemius-plantaris-soleus (GPS) muscle group in situ in AK1 Ϫ/Ϫ mice and wild-type (WT) controls over a range of contraction frequencies (30 -120 tetani/min). We found that AK1 Ϫ/Ϫ muscle exhibited a diminished inosine 5Ј-monophosphate formation rate (14% of WT) and an inordinate accumulation of ADP (ϳ1.5 mM) at the highest energy demands, compared with WT controls. AK-deficient muscle exhibited similar initial contractile performance (521 Ϯ 9 and 521 Ϯ 10 g tension in WT and AK1 Ϫ/Ϫ muscle, respectively), followed by a significant slowing of relaxation kinetics at the highest energy demands relative to WT controls. This is consistent with a depressed capacity to sequester calcium in the presence of high ADP. However, the overall pattern of fatigue in AK1 Ϫ/Ϫ mice was similar to WT control muscle. Our findings directly demonstrate the importance of AMP formation and subsequent deamination in limiting ADP accumulation. Whole muscle contractile performance was, however, remarkably tolerant of ADP accumulation markedly in excess of what normally occurs in skeletal muscle. AMP deaminase; tetanic contraction; muscle relaxation; calcium handling; cross-bridge cycling SKELETAL MUSCLE can maintain favorable energetic conditions in the face of great energetic demands. For example, the ATP consumption rate needed to support a tetanic contraction (ϳ10 mol/g per second) is ϳ200-fold above the resting rate in rat fast twitch fibers (28, 42). The metabolic challenge this represents is highlighted by the fact that the ATP content of mammalian skeletal muscle is only 6 -7 mol/g. Therefore, without compensation, the entire ATP pool would be completely depleted in Ͻ1 s at this rate. Obviously, this does not occur due to the ATP synthetic processes of oxidative phosphorylation, glycolysis, the creatine kinase (CK) reaction, and the adenylate kinase (AK) reaction. Moreover, the free energy available from ATP hydrolysis (⌬G ATP ) is a function of the ATP content relative to the products of ATP hydrolysis, namely ADP and inorganic phosphate (P i )where ⌬G°A TP is the conventional expression for the free engery of ATP at standard conditions, R is the ideal gas constant at 0.0083143 kJ/mol⅐K, and T is the temperature in Kelvin. Furthermore, the accumulation of ADP and P i direct physiological consequences independent of an impaired ⌬G ATP . Thus, the metabolic challenge when ATP turnover is high, is to preserve ATP content and limit inordinate accumulation of ADP and P i .When the rate of ATP hydrolysis is out of balance with the rate of ATP synthesis, the need to limit the accumulation of ADP and P i is the greatest. The rea...

letion of the major adenylate kinase AK1 isoform, which catalyzes adenine nucleotide exchange, disrupts cellular energetic economy and compromises metabolic signal transduction. However, the consequences of deleting the AK1 gene on cardiac energetic dynamics and performance in the setting of ischemia-reperfusion have not been determined. Here, at the onset of ischemia, AK1 knockout mice hearts displayed accelerated loss of contractile force compared with wild-type controls, indicating reduced tolerance to ischemic stress. On reperfusion, AK1 knockout hearts demonstrated reduced nucleotide salvage, resulting in lower ATP, GTP, ADP, and GDP levels and an altered metabolic steady state associated with diminished ATP-to-P i and creatine phosphate-to-Pi ratios. Postischemic AK1 knockout hearts maintained ϳ40% of ␤-phosphoryl turnover, suggesting increased phosphotransfer flux through remaining adenylate kinase isoforms. This was associated with sustained creatine kinase flux and elevated cellular glucose-6-phosphate levels as the cellular energetic system adapted to deletion of AK1. Such metabolic rearrangements, along with sustained ATP-to-ADP ratio and total ATP turnover rate, maintained postischemic contractile recovery of AK1 knockout hearts at wild-type levels. Thus deletion of the AK1 gene reveals that adenylate kinase phosphotransfer supports myocardial function on initiation of ischemic stress and safeguards intracellular nucleotide pools in postischemic recovery. energy metabolism; adenine nucleotides; glycolysis; phosphotransfer; oxygen-18 phosphoryl labeling; phosphorus-31 nuclear magnetic resonance MAINTENANCE OF OPTIMAL CARDIAC function requires precise control of cellular nucleotide ratios and high-energy phosphoryl fluxes (11,22,30,32,33,36,40). Within the cellular energetic infrastructure, adenylate kinase has been recognized as an important phosphotransfer enzyme that catalyzes adenine nucleotide exchange (ATP ϩ AMP ª 2ADP) and facilitates transfer of both ␤-and ␥-phosphoryls in ATP (9,15,24,25,43). In this way, adenylate kinase doubles the energetic potential of ATP as a high-energy-phosphoryl carrying molecule and provides an additional energy source under conditions of increased demand and/or compromised metabolic state (13-15, 31, 35, 42). By regulating adenine nucleotide processing, adenylate kinase has been implicated in metabolic signal transduction (12,15,27). Indeed, phosphoryl flux through adenylate kinase has been shown to correlate with functional recovery in the metabolically compromised heart (30) and to facilitate intracellular energetic communication (3,9,13